Formulation comprising extracellular vesicles, method for producing the same, and uses thereof

ABSTRACT

Disclosed herein is a formulation comprising an extracellular vesicle (EV), and a therapeutic active agent induced or embedded in the EV. According to preferred embodiments of the present disclosure, the EV is isolated from umbilical cord mesenchymal stem cells, and the active agent may be a growth factor, an immune-modulating agent, a small molecule, an siRNA, cDNA or a plant ingredient; for example, curcumin. Also disclosed herein are methods for producing the present formulation, and uses of the present formulation in the treatment of various diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/018,055, filed Jun. 26, 2018, which claims the benefit of U.S.Provisional Application No. 62/524,624, filed Jun. 26, 2017; the contentof the application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present disclosure in general relates to the field of a biologicaldelivery system. More particularly, the present disclosure relates to anextracellular vesicle (EV) formulation comprising one or more activeagents, and the uses thereof.

2. Description of Related Art

Extracellular vesicle (EV) is a cell-derived membrane vesicle, andrepresents an endogenous mechanism for intercellular communication. Ingeneral, a cell (i.e., a donor cell) may release different types of EVs,including exosome (with diameter ranging from 50 to 150 nm), andmicrovesicle (with diameter ranging from 50 nm to 500 nm). The releasedEVs can be up-taken by a recipient cell via endocytosis, membrane fusionor specific ligand-receptor internalization, and thereby resulted indelivering the content of EVs (e.g., the nucleic acid,lipopolysaccharide, protein and/or lipid from the donor cell) to therecipient cells.

EVs can be isolated from various sources of biofluids, such as blood,urine, cerebrospinal fluid, malignant ascites, breast milk,bronchoalveolar lavage and saliva. Since the content of EVs reflects thestatus of the donor cell, it may be used as a biomarker in theapplication of diagnosis, prognosis, and epidemiology. For example, EVsderived from cancer cells or released from nearby stromal cells orleukocytes may be released into circulation for guiding cancer cellinvasion and metastasis; vice versa, mesenchymal stem cells and/orimmune cells may release EVs in response to cancer cells for antagonismof cancer cell invasion and metastasis. Accordingly, the EVs isolatedfrom ascites is useful for diagnosing ovarian cancer and determining thestage of the diseases; whereas the EVs isolated from blood is useful fordiagnosing ovarian cancer and breast cancer.

EV is also a suitable candidate for organelle therapy, based on theadvantage of its lipid bilayer properties. EVs are capable of directingcell fusion, carrying proteins or miRNAs for cell-cell communicationsand even reaching distal organ such as kidney, lung and central nervoussystem via crossing the major biological membranes.

EV is also a suitable candidate for drug delivery, based on theadvantage of high biocompatible, low immunogenicity, nanoparticle size,and capable of crossing the major biological barriers (includingblood-brain barrier (BBB)). However, the therapeutic effect usuallyvaries with the factors, such as disease, disease severity and the EVproperty (e.g., the cellular origin, the size and the content of EVs).Formulations of EVs with pre-conditional culture stimulations, or withpost-isolation incorporation of small molecules, peptides, nucleicacids, aptamers or scaffolds may improve and expand the therapeuticapplications.

In view of the foregoing, there exists in the related art a need for amodified EV that can be wildly applied in the treatment of variousdiseases, such as inflammatory disease, degenerative disease, infectiousdisease, metabolic disease, dysplastic disease, diabetes and cancer.

SUMMARY

The following presents a simplified summary of the disclosure in orderto provide a basic understanding to the reader. This summary is not anextensive overview of the disclosure and it does not identifykey/critical elements of the present invention or delineate the scope ofthe present invention. Its sole purpose is to present some conceptsdisclosed herein in a simplified form as a prelude to the more detaileddescription that is presented later.

One aspect of the present disclosure is directed to a formulation, whichcomprises an EV and one or more active agent encapsulated in the EV.According to embodiments of the present disclosure, the EV is derivedfrom a mesenchymal stem cell. Preferably, the mesenchymal stem cell isisolated from Wharton jelly of umbilical cord.

The active agent is selected from the group consisting of, a growthfactor, an immune-modulating agent, an anti-cancer agent, ananti-inflammatory agent, an anti-infection agent, an anti-aging agent,an anti-oxidant agent, an anti-radiation agent, a nucleic acid and acombination thereof. According to some embodiments of the presentdisclosure, the active agent is aspirin, bryostatin or curcumin.According to other embodiments, the active agent is the nucleic acidselected from the group consisting of, small interference ribonucleicacid (siRNA), small hairpin ribonucleic acid (shRNA), andmicro-ribonucleic acid (miRNA). According to alternative embodiments ofthe present disclosure, the active agent is a polypeptide, whichmodulates T cell differentiation. Additionally or alternatively, theactive agent may be an anti-oxidant agent, a biological agent, or ananti-aging agent such as metformin, β-lapachone etc.

According to certain embodiments, the present formulation is prepared bya method comprising the steps of,

(a-1) isolating the EV from the mesenchymal stem cell; and

(b-1) mixing the isolated EV of step (a-1) and the active agent so as toencapsulate the active agent in the isolated EV.

Alternatively, the present formulation is prepared by a methodcomprising the steps of,

(a-2) subjecting the mesenchymal stem cell to the treatment of theactive agent; and

(b-2) isolating the EV from the active agent treated mesenchymal stemcell of step (a-2).

Still alternatively, the present formulation is prepared by a methodcomprising the steps of,

(a-3) irradiating the mesenchymal stem cell with a linear accelerator,X-ray or gamma ray; and

(b-3) isolating the EV from the irradiated mesenchymal stem cell of step(a-3).

According to some embodiments of the present disclosure, in the step(a), the mesenchymal stem cell is irradiated with X-ray at a dose of1-10 Gray (Gy). More preferably, the mesenchymal stem cell is irradiatedwith X-ray at a dose of 1-5 Gy. According to one working example, themesenchymal stem cell is irradiated with X-ray at a dose of 3 Gy.

Another aspect of the present disclosure pertains to a method oftreating an inflammatory disease, dry eye, degenerative disease, cancer,infectious disease and/or aging in a subject. The method comprisesadministering to the subject an effective amount of the presentformulation.

Non-limiting examples of the inflammatory diseases include psoriasis,colitis, burn injury, acute kidney injury, traumatic brain injury, skininjury, arthritis and autoimmune diseases.

The degenerative diseases treatable with the present method include, butare not limited to, Parkinson's disease, Alzheimer's disease, dementia,stroke, chronic kidney disease, chronic lung disease and hearing loss.

The cancer treatable with the present method may be any of gastriccancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer,renal cancer, colorectal cancer, cervical cancer, ovarian cancer, braintumor, prostate cancer, hepatocellular carcinoma, melanoma, esophagealcarcinoma, multiple myeloma, or head and neck squamous cell carcinoma.

Regarding the infectious disease, it may be caused by a bacterium, virusor fungus.

In general, the formulation disclosed herein may be administered to thesubject by a suitable route. Non-limiting examples of the suitable routeinclude, topical, mucosal (e.g. intraconjunctival, intranasal,intratracheal), oral, intraspinal (e.g. intrathecal), intravenous,intraarterial, intramuscular, subcutaneous, intraarticular,intraventrical, intracerebroventricular, intraperitoneal injection andintra-middle ear administration.

The subject is a mammal; preferably, a human.

Many of the attendant features and advantages of the present disclosurewill becomes better understood with reference to the following detaileddescription considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present description will be better understood from the followingdetailed description read in light of the accompanying drawings, where:

FIG. 1 is the data of flow cytometry that depicts the expression ofcellular marker on umbilical cord mesenchymal stem cells (ucMSCs)according to one embodiment of the present disclosure.

FIG. 2 is the data depicting the isolation and verification ofucMSC-naive-EVs (i.e., EVs derived from ucMSC without pre-treatment)according to Example 1 of the present disclosure. Panel A: the result offlow cytometry depicting the expression level of CD81 marker onucMSC-naive-EVs. Panel B: the result of western blot depicting theexpression level of CD63 marker on ucMSC-naive-EVs. Panel C: the resultof transmission electron microscopy (TEM) depicting the morphology ofucMSC-naive-EVs, in which the diameter of ucMSC-naive-EVs was about50-150 nm. Scale bar represents 0.1 um. Panel D: EVs promote the growthof C2C12 myoblasts in a dose-dependent manner, in which compared withthe EVs derived from DLD-1 cancer cells (designated as DEV), theucMSC-naive-EVs (designated as MEV) exhibited a significant effect onenhancing cellular growth.

FIG. 3 is a histogram depicting the effect of specified EVs on thegrowth of muscle cell according to Example 2 of the present disclosure.EV: the C2C12 myoblasts treated with ucMSC-naive-EVs; EV-Cur: the C2C12myoblasts treated with EV-curcumin (i.e., EV having curcuminencapsulated or embedded therein); Bryo-EV: the C2C12 myoblasts treatedwith bryostatin-EV (i.e., EV isolated from the culture supernatant ofucMSCs pretreated with bryostain for 24 hours); Asp-EV: the C2C12myoblasts treated with aspirin-EV (i.e., EV isolated from the culturesupernatant of ucMSCs pretreated with aspirin for 24 hours). *denotesP<0.05 as analyzed by Mann Whitney U test.

FIG. 4 is a histogram depicting the synergistic effect of specifiedtreatment on anti-inflammation response according to Example 2 of thepresent disclosure, in which the EVs comprising curcumin therein(EV-Curcumin) significantly decreased the reactive oxygen species incells. Data shown were calculated from 3 triplicate experiments andstatistically analyzed by Mann Whitney U test.

FIG. 5 is the data depicting the miRNA profiles of DLD-1-derived EVs(DEVs), ucMSC-naive-EVs (MEV), or EV-curcumin according to Example 3 ofthe present disclosure, in which 36 out of 2,000 Homo sapiens miRNAs(hsa miRs) were differently expressed in these EVs. Compared withucMSC-naive-EV, the incorporation of curcumin increased the expressionof 5 miRNAs (marked in red color) and decreased the expression of 2miRNAs (marked in green color).

FIG. 6 is the data depicting the protein profiles of DLD-1-derived EVs(DEVs), ucMSC-naive-EVs (MEV), or EV-curcumin according to Example 3 ofthe present disclosure, in which the corporation of curcumin increasedthe expression of 6 proteins and decreased the expression of 4 proteins,while defining the increase over 4 fold and the decrease under 1/4 fold.

FIG. 7 is the data of EVs tracking analysis that depicts theconcentration and size of specified EVs according to Example 2 of thepresent disclosure. Naïve EVs show a size of mode at 144 nm, EVsincorporating curcumin after isolation (EV-curcumin) reveal a smallersize of mode, EVs derived from pre-culture with bryostatin(bryostatin-EV) show a larger size and EVs derived from preculture withaspirin show a larger size and the highest concentration.

FIG. 8 is the data depicting the effect of specified EVs on regulating Tcell differentiation according to Example 2 of the present disclosure.Panel A: the expression level of CXCR3, a cellular marker of Th1 cells;*denotes P<0.05 between neg group and Ctrl group. Panel B: theexpression level of CCR4, a cellular marker of Th2 cells; *denotesP<0.05 between Ctrl group and EV group; # denotes P<0.05 between byro-EVgroup and EV-10a group. Panel C: the expression level of CD25, acellular marker of regulatory T (Treg) cells; *denotes P<0.05 betweenCtrl group and EV group; # denotes P<0.05 between byro-EV group andEV-10a group. Panel D: the expression level of CCR6, a cellular markerof Th17 cells; *denotes P<0.05 between byro-EV group and EV-10a group.neg: PBS treatment only; Ctrl: the treatment of anti-CD3 and anti-CD28antibodies; EV: the treatment of anti-CD3 and anti-CD28 antibodies inthe presence of ucMSC-naive-EV; byro-EV: the treatment of anti-CD3 andanti-CD28 antibodies in the presence of bryostatin-EV; Asp-EV: thetreatment of anti-CD3 and anti-CD28 antibodies in the presence ofaspirin-EV; EV-10a: the treatment of anti-CD3 and anti-CD28 antibodiesin the presence of EV-miRNA10a (i.e., EV having miRNA10a encapsulated orembedded therein).

FIG. 9 is a histogram depicting the protecting effect of EVs onirradiation-induced reactive oxygen species (ROS) production accordingto Example 4 of the present disclosure, in which the EVs wereucMSC-irradiation-EV (i.e., the EVs isolated from ucMSCs irradiated withor without 3 Gy (sublethal dose) delivered by a linear accelerator in asingle fraction). Ctrl: the ucMSCs treated with PBS and irradiated with0 or 6 Gy of X-ray; EVs: the ucMSCs treated with ucMSCs-naive-EVs andirradiated with 0 or 6 Gy of X-ray; Rad-EV: the ucMSCs treated withucMSC-irradiation-EV and irradiated with 0 or 6 Gy of X-ray. The Pvalues are calculated by Mann Whitney U test. *denotes P<0.05 between 0Gy irradiation and 6 Gy irradiation of Ctrl group. # denotes P<0.05between 6 Gy irradiation of EVs group and 6 Gy irradiation of Rad-EVgroup.

FIG. 10 is the data depicting the effect of specified treatment on Tcell differentiation according to Example 5 of the present disclosure.Panel A: the expression of CCR4 on CD4⁺ T cells; *denotes P<0.05 betweenNeg group and Ctrl group. Panel B: the expression of CCR4 on CD8⁺ Tcells; *denotes P<0.05 between Ctrl group and EV group; # denotes P<0.05between EV group and EV-ZIP group. Neg: PBS treatment only; Ctrl: thetreatment of anti-CD3 and anti-CD28 antibodies; EV: the treatment ofanti-CD3 and anti-CD28 antibodies in the presence of ucMSC-naive-EV;ZIP: the treatment of polypeptide of SEQ ID NO: 1; EV-ZIP: the treatmentof EV-ZIP.

FIG. 11 is a histogram depicting the anti-migration effect of specifiedEVs on cancer cells according to Example 6 of the present disclosure.Ctrl/DLD-1: the DLD-1 cells treated with PBS; DEV/DLD-1: the DLD-1 cellstreated with DLD-1-derived EVs; MEV/DLD-1: the DLD-1 cells treated withucMSC-naive-EV; DEV-miR-10a/DLD-1: the DLD-1 cells treated with EVs,which was derived from DLD-1 cells and comprised miRNA10a therein;MEV-miR-10a/DLD-1: the DLD-1 cells treated with EV-miRNA10a. *denotes byP<0.05 between DEV/DLD-1 group and MEV/DLD-1 group. # denotes by P<0.05between DEV/DLD-1 group and DEV-miR-10a/DLD-1 group.

FIG. 12 is the data depicting the therapeutic effect of specifiedtreatment on psoriasis in a mouse model according to Example 7 of thepresent disclosure. Panels A and B are respectively the photographs andquantified data of ear auricle thickness. Control: the mice treated withPBS only; Vehicle: the mice administrated with imiquimod followed by thetreatment of PBS; EV: the mice administrated with imiquimod followed bythe treatment of ucMSC-naive-EV; EV-curcumin: the mice administratedwith imiquimod followed by the treatment of EV-curcumin. *denotes P<0.05as tested by Mann Whitney U test; compared with the control group.

FIG. 13 is data depicting the therapeutic effect of specified treatmenton dry eye in a mouse model according to Example 7 of the presentdisclosure. Panel A: photographs depicting the conjunctiva of micereceived vehicle or specified treatment. Panel B: the dry eye score ofmice treated with serum-free medium (SFM) or ucMSC-naive-EV (EV). PanelC: the dry eye score of mice treated with serum-free medium (SFM) oraspirin-EV. O.D.: right eye; O.S.: left eye. *denotes P<0.05 as analyzedby Mann Whitney U test; compared with SFM group.

FIG. 14 is data depicting the protecting effect of specified treatmenton hearing loss in a mouse model according to Example 7 of the presentdisclosure. Panels A and B: the auditory brain response (ABR) results ofmice receiving specified treatment. Panel C: the immunofluorescenceimages of organ of Corti explants, in which arrows indicated the loss ofhair cells after treatment. Scale bar represents 50 μm. Vehicle: themice administrated with cisplatin followed by the treatment of PBS; EV:the mice administrated with cisplatin followed by the treatment ofucMSC-naive-EV; EV-curcumin: the mice administrated with cisplatinfollowed by the treatment of EV-curcumin; curcumin: the miceadministrated with cisplatin followed by the treatment of curcumin.

FIG. 15 is the data depicting the anti-aging effect of specifiedtreatment in a mouse model according to Example 7 of the presentdisclosure. Panel A: Escape latency of mice administered with or withoutD-galactose. Panel B: Escape latency of mice receiving specifiedtreatments.

FIG. 16 is the data depicting the distribution of ucMSC-naive-EV in thespinal cord according to Example 7 of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description provided below in connection with the appendeddrawings is intended as a description of the present examples and is notintended to represent the only forms in which the present example may beconstructed or utilized. The description sets forth the functions of theexample and the sequence of steps for constructing and operating theexample. However, the same or equivalent functions and sequences may beaccomplished by different examples.

I. Definition

For convenience, certain terms employed in the specification, examplesand appended claims are collected here. Unless otherwise defined herein,scientific and technical terminologies employed in the presentdisclosure shall have the meanings that are commonly understood and usedby one of ordinary skill in the art. Also, unless otherwise required bycontext, it will be understood that singular terms shall include pluralforms of the same and plural terms shall include the singular.Specifically, as used herein and in the claims, the singular forms “a”and “an” include the plural reference unless the context clearlyindicates otherwise. Also, as used herein and in the claims, the terms“at least one” and “one or more” have the same meaning and include one,two, three, or more.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in therespective testing measurements. Also, as used herein, the term “about”generally means within 10%, 5%, 1%, or 0.5% of a given value or range.Alternatively, the term “about” means within an acceptable standarderror of the mean when considered by one of ordinary skill in the art.Other than in the operating/working examples, or unless otherwiseexpressly specified, all of the numerical ranges, amounts, values andpercentages such as those for quantities of materials, durations oftimes, temperatures, operating conditions, ratios of amounts, and thelikes thereof disclosed herein should be understood as modified in allinstances by the term “about”. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the present disclosureand attached claims are approximations that can vary as desired. At thevery least, each numerical parameter should at least be construed inlight of the number of reported significant digits and by applyingordinary rounding techniques.

The term “formulation” as used herein refers to an composition includingone or more phospholipids, and one or more active agents encapsulated orembedded by said one or more phospholipids. The formulation of thepresent disclosure comprises an EV, which has the structure of aliposome (i.e., a hydrophilic core enclosed by a lipid bilayer), and atleast one active agent encapsulated in the liposome structure of EV.

The term “encapsulate” refers to the process of encompassing, encasing,or otherwise associating two or more materials such that theencapsulated material is immobilized within or onto the encapsulatingmaterial. The term “encapsulate” as used herein refers to the inclusionof one or more molecule (e.g., the present active agent) in a hollowparticle, for example, in the hydrophilic core of EV. In general, theactive agent may be surrounded or partially surrounded by the lipidbilayer of EV.

As used herein, the term “Wharton's jelly”, also known as inter-laminarjelly, is a subset of the umbilical cord matrix, and refers to amucous-connective tissue substance found in the umbilical cord. Thecomponents of Wharton's Jelly include a mucous connective tissue, whichcomprises myofibroblasts, fibroblasts, macrophages, mesenchymal stemcells and an amorphous ground substance composed of hyaluronic acid andpossibly other as yet uncharacterized cell populations. Wharton's Jellyis one component of the umbilical cord.

The term “effective amount” as referred to herein designate the quantityof a component, which is sufficient to yield a desired response. Fortherapeutic purposes, the effective amount is also one in which anytoxic or detrimental effects of the component are outweighed by thetherapeutically beneficial effects. The specific effective or sufficientamount will vary with such factors as the particular condition beingtreated, the physical condition of the patient (e.g., the patient's bodymass, age, or gender), the type of mammal or animal being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulations employed and the structure of thecompounds or its derivatives. Effective amount may be expressed, forexample, in grams, milligrams or micrograms or as milligrams perkilogram of body weight (mg/Kg). Alternatively, the effective amount canbe expressed in the concentration of the active component (e.g., theformulation of the present disclosure), such as molar concentration,mass concentration, volume concentration, molality, mole fraction, massfraction and mixing ratio. Specifically, the term “therapeuticallyeffective amount” used in connection with the formulation describedherein refers to the quantity of the formulation, which is sufficient toalleviate or ameliorate the symptoms associated with the inflammatorydisease, degenerative disease, infectious disease, metabolic disease,dysplastic disease, diabetes or cancer in the subject. Persons havingordinary skills could calculate the human equivalent dose (HED) for themedicament (such as the present formulation) based on the dosesdetermined from animal models. For example, one may follow the guidancefor industry published by US Food and Drug Administration (FDA) entitled“Estimating the Maximum Safe Starting Dose in Initial Clinical Trialsfor Therapeutics in Adult Healthy Volunteers” in estimating a maximumsafe dosage for use in human subjects.

The term “subject” refers to a mammal including the human species thatis treatable with methods of the present invention. The term “subject”is intended to refer to both the male and female gender unless onegender is specifically indicated.

II. DESCRIPTION OF THE INVENTION

The objective of the present disclosure aims at providing a formulationcomprising an EV and one or more active agent, methods of producing theformulation, the uses thereof.

(I) The Present Formulation

The first aspect of the present disclosure is directed to a formulation,which comprises an EV derived from a mesenchymal stem cell (MSC), which,in term, is isolated from Wharton jelly; and one or more active agentencapsulated therein.

EV is a spherical vesicle having the structure of a liposome, i.e., ahydrophilic core enclosed by a lipid bilayer; thus is suitable as ameans for drug delivery, in which active agent(s) may be encapsulated inthe liposome structure. Depending on desired effects, the active agentsuitable for encapsulating in the EV liposome structure may be any of agrowth factor, an immune-modulating agent, an anti-cancer agent, ananti-inflammatory agent, an anti-infection agent, an anti-aging agent,an anti-oxidant agent, an anti-radiation agent, a vitamin, a heat-shockprotein (HSP), a nucleic acid, a plant ingredient, or a combinationthereof.

Compared with the EV derived from other tissues/cells, the EV derivedfrom umbilical cord mesenchymal stem cells (ucMSCs) is advantageous inat least the five following aspects:

(1) Similar to umbilical cord blood, in which different factors derivedfrom stem cells or serum have been shown to rescue animals with aging.The EVs isolated from ucMSCs comprise higher levels of growth factors,including placental growth factor (PLGF) and granulocytecolony-stimulating factor (G-CSF).

(2) According to proteomic analysis, the expression level ofproliferative molecules is higher in ucMSCs than in adult type MSCs.

(3) Compared with adult type MSCs, ucMSCs possess longer telomeres. Theproliferation capability renders ucMSCs suitable for large-scalepreparation of EVs.

(4) EVs derived from ucMSCs can be treated with irradiation ormodulators so as to prepare desired formulations for the treatment ofregenerative, anti-infectious or anti-inflammatory disorders, or producea radio-protecting effect.

(5) EVs possess lipid bilayer property, which can be loaded withpeptides or nucleic acids, such as DNA aptamers, RNA aptamers, miRNA,siRNA and etc.

(6) The EVs may be prepared as an anti-alkaline, anti-acid, oranti-freeze formulation via integrating desired compounds (e.g., thehydrophobic or hydrophilic compound) in the liposome structure thereof.

According to certain embodiments of the present disclosure, the activeagent is a plant ingredient extracted from a plant component selectedfrom the group consisting of leaf, stalk, root, flower, tuber, pollen,fruit and seed. Alternatively, the active agent may be an ingredientextracted from a fungus or bacteria. According to certain examples, theactive agent is aspirin, lipopolysaccharide, quercetin, curcumin,astragalus, resveratrol, astaxathin or polyphenol. In one workingexample of the present disclosure, the active agent is curcumin.

According to some embodiments of the present disclosure, the activeagent is the growth factor. Non-limiting examples of the growth factorinclude, but are not limited to, angiopoietin, macrophagecolony-stimulating factor (M-CSF), granulocyte colony-stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF),placental growth factor (PLGF), vascular endothelial growth factor(VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF),one morphogenetic protein (BMP), endoglin, endothelin, leptin,follistatin, hepatocyte growth factor (HGF), insulin-like growth factor(IGF), keratinocyte growth factor (KGF), nerve growth factor (NGF),growth factor-α (TGF-α), transforming growth factor-β (TGF-β), cartilagegrowth factor (CGF), stem cell factor (SCF), brain-derived neurotrophicfactor (BDNF), platelet-derived growth factor (PDGF), interleukin (IL)and ephrin. In one working example of the present disclosure, the activeagents are angiopoietin, endoglin, FGF-2, HGF, IL-8 and VEGF-C.

Exemplary anti-cancer drugs include, but are not limited to, curcumin,interferons, cytokines (e.g., tumor necrosis factor, interferon α,interferon γ), antibodies (e.g. Herceptin (trastuzumab), T-DM1, AVASTIN(bevacizumab), ERBITUX (cetuximab), Vectibix (panitumumab), Rituxan(rituximab), and Bexxar (tositumomab)), anti-estrogens (e.g. tamoxifen,raloxifene, and megestrol), LHRH agonists (e.g. goscrclin andleuprolide), anti-androgens (e.g. flutamide and bicalutamide),photodynamic therapies (e.g. vertoporfin (BPD-MA), phthalocyanine,photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogenmustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil,estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) andlomustine (CCNU)), alkylsulphonates (e.g. busulfan and treosulfan),triazenes (e.g. dacarbazine, temozolomide), platinum containingcompounds (e.g. cisplatin, carboplatin, oxaliplatin), vinca alkaloids(e.g. vincristine, vinblastine, vindesine, and vinorelbine), taxoids(e.g. paclitaxel or a paclitaxel equivalent such as nanoparticlealbumin-bound paclitaxel (Abraxane), docosahexaenoic acidbound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamatebound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103,XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound tothree molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to theerbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel,e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel,taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors(e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMPdehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin,and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea anddeferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine,doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosineanalogs (e.g. cytarabine (ara C), cytosine arabinoside, andfludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin A analogs, Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH1060), vitamin K, isoprenylation inhibitors (e.g. lovastatin),dopaminergic neurotoxins (e.g. 1-methyl-4-phenylpyridinium ion), cellcycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D,dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin),anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomaldoxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin,mitoxantrone), MDR inhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors(e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosinekinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606),cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825),erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®,CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib(CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib(semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib(PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK),trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®),cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®),nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®),alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus(TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258,CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066,PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534,JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib(AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), proteasomeinhibitors (e.g., bortezomib (Velcade)), mTOR inhibitors (e.g.,rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus,AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226(Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980(Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen,gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide,dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin,plicamycin, asparaginase, aminopterin, methopterin, porfiromycin,melphalan, leurosidine, leurosine, chlorambucil, trabectedin,procarbazine, discodermolide, carminomycin, aminopterin, and hexamethylmelamine.

Examples of anti-inflammatory molecule include, but are not limited tocurcumin, non-steroidal anti-inflammatory drugs (NSAIDs) including,alclofenac, alclometasone dipropionate, algestone acetonide, alphaamylase, amcinafal, amcinafide, amfenac sodium, amiprilosehydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazidedisodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains,broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen,clobetasol propionate, clobetasone butyrate, clopirac, cloticasonepropionate, cormethasone acetate, cortodoxone, decanoate, deflazacort,delatestryl, depo-testosterone, desonide, desoximetasone, dexamethasonedipropionate, diclofenac potassium, diclofenac sodium, diflorasonediacetate, diflumidone sodium, diflunisal, difluprednate, diftalone,dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium,epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen,fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, flazalone,fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin,flunixin meglumine, fluocortin butyl, fluorometholone acetate,fluquazone, flurbiprofen, fluretofen, fluticasone propionate,furaprofen, furobufen, halcinonide, halobetasol propionate, halopredoneacetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol,ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole,intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen,lofemizole hydrochloride, lomoxicam, loteprednol etabonate,meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate,mefenamic acid, mesalamine, meseclazone, mesterolone,methandrostenolone, methenolone, methenolone acetate, methylprednisolonesuleptanate, momiflumate, nabumetone, nandrolone, naproxen, naproxensodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin,oxandrolane, oxaprozin, oxyphenbutazone, oxymetholone, paranylinehydrochloride, pentosan polysulfate sodium, phenbutazone sodiumglycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicamolamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone,proxazole, proxazole citrate, rimexolone, romazarit, salcolex,salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin,stanozolol, sudoxicam, sulindac, suprofen, talmetacin, talniflumate,talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam,tesimide, testosterone, testosterone blends, tetrydamine, tiopinac,tixocortol pivalate, tolmetin, tolmetin sodium, triclonide,triflumidate, zidometacin, and zomepirac sodium. In addition, cytokineantagonists, such as aptamers of IL-10, IL-6, IL-8, TNF-alpha (TNF-α),IL-5, IL-13, TGF-beta (TGF-β), VEGF and etc., may be used foranti-inflammatory effects.

Non-limiting examples of anti-oxidant agents include amine (e.g.,N,N-diethylhydroxylamine, and amino-guanidine), arginine pilolate,ascorbic acid and its salts, ascorbyl ester of fatty acid, bioflavonoid,butylated hydroxy benzoic acid and its salt, dihydroxy fumaric acid andits salts, gallic acid and its alkyl esters (e.g., propyl gallate, anduric acid), glycine pidolate,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, lipoic acid,lysine, melanin, methionine, nordihydroguaiaretic acid, proline,silymarin, sorbic acid and its salts, sulfhydryl compounds (e.g.,glutathione), superoxide dismutase, catalase, tea extract, grapeskin/seed extract, rosemary extract, tocopherol acetate, tocopherol,tocopherol sorbate and the combination thereof. These incorporations canbe loaded into a scaffold for better tissue localization.

Examples of anti-aging agent include, but are not limited to, curcumin,coenzyme Q10, xanthophyll (e.g., astaxanthin, fucoxanthin andzeaxanthin), L-glutathione, retinoid, α-hydroxyl acids, β-hydroxyl acidand lutein. Alternatively, the anti-aging agent may be a proteoglycan, aglycoprotein or a glycolipid, which is optionally loaded into a scaffoldfor better tissue localization.

Examples of anti-infectious agent include, but are not limited to, LL37,interferon alpha, hydrophilic and hydrophobic antibiotics, and aptamers.

The vitamin is any of vitamin A, vitamin D, vitamin K or vitamin E.

Non-limiting examples of HSP include, but are not limited to, HSP10,HSP27, HSP40, HSP60, HSP70, HSP72, HSP90, HSP100 and HSP104.

The nucleic acid may be a DNA or RNA. According to certain embodimentsof the present disclosure, the nucleic acid is an RNA, for example, ansiRNA, an shRNA or an miRNA. Alternatively, the nucleic acid may be aDNA aptamer, an RNA aptamer, or an affixmer. Non-limiting examples ofthe miRNA include agomirs (such miRNA125a, miRNA21, miRNA214, miRNA410,miRNA495 and 548L-5p etc.), and antagomirs (such as miRNA10a,miRNA182-5p, miRNA155a etc.). In one working example of the presentdisclosure, the nucleic acid is miRNA10a, which comprises the nucleotidesequence of SEQ ID NO: 2.

Alternatively, the active agent of the present formulation may be anasta-carotenoid selected from the group consisting of, astaxanthin,carotene, echinenone, canthaxanthin, adonirubin, doradexanthin,zeaxanthin, antheraxanthin, violaxanthin, neoxanthin and a combinationthereof.

Non-limiting examples of immune-modulating agent include, interleukin(such as IL-2, IL-7, IL-12), cytokine (such as granulocytecolony-stimulating factor (G-CSF) and interferon (IFN)), chemokine (suchas CXCL13, CCL26 and CXCL7), antagonist of immune checkpoint (such asanti-CTLA-4, anti-PD1 or anti-PD-L1 (ligand of PD-1), anti-LAG3,anti-B7-H3, synthetic cytosine phosphate-guanosine (CpG)oligodeoxynucleotide, and glucan), and modulator of Tregs (such ascyclophosphamide). In one specific example of the present disclosure,the immune-modulating agent is a polypeptide comprising the amino acidsequence of SEQ ID NO: 1.

In some embodiments, the active agent is aspirin, lipopolysaccharide,metformin, peptides or bryostatin.

The produced formulation is stable to freeze condition (e.g., beingstable for at least three months in liquid or freeze dried form), acidtreatment (e.g., the formulation comprising astaxathin), and alkalinetreatment (e.g., the formulation comprising curcumin). In addition, thepresent formulation is also stable in polyols, such as glycerol andpolyethylene glycol (PEG).

(II) Production of the Present Formulation

The active agent may be incorporated into the liposome structure of EVsvia a pre-conditional treatment (e.g., incubating the active agent withthe MSCs, or treating the MSCs with an irradiation or a small moleculesuch as aspirin, metformin, lipopolysaccharide, or resveratrol) or apost-isolation treatment (e.g., incorporating the active agent such asastaxathin or curcumin, with EVs derived from MSCs treated with orwithout irradiation). The present disclosure provides three exemplarymethods for the preparation the present EV formulation.

The first method comprises the steps of,

(a-1) isolating EVs from a mesenchymal stem cell (MSC); and

(b-1) mixing the isolated EVs of step (a-1) and an active agent so as toencapsulating the active agent therein.

In general, EVs may be isolated from suitable sources, preferably, fromMSCs in accordance with process well known in the related art. Examplesof method suitable for such purpose include, but are not limited to,differential centrifugation, sucrose gradient centrifugation,microfiltration, antibody-coated magnetic bead, bioreactor andmicrofluidic device. Alternatively, EVs may be isolated fromtissues/biofluids with the aid of suitable commercial kit, such as,EXOQUICK.

According to embodiments of the present disclosure, EVs are obtainedfrom the culture supernatant of MSCs. Generally, MSCs are cultured inlow glucose DMEM with 10% fetal bovine serum (FBS) followed by thetreatment of a serum-free medium to induce the secretion of EVs fromMSCs to the supernatant; the secreted EVs are then harvested by anysuitable means. In one preferred embodiment, the secreted EVs in thesupernatant are isolated by microfiltration, in which particles rangingfrom 0.02 to 0.20 μm are retained.

Alternatively, the hollow-fiber bioreactor (a three-dimensional cellculturing system) is used for the purpose of improving the EV productionefficiency. The bioreactor is a compact system, in which a pump forcesmedium through a cartridge containing cell cultures (e.g., MSC cellculture), and tiny hollow fibers mediate the exchanges of nutrient andwaste. The EVs produced by the cells (e.g., MSCs) secreting to thehollow fibers are captured in the medium and harvested by any suitablemeans.

To produce the present formulation, in the step (b-1), the isolated EVsof step (a-1) and active agents are mixed for sufficient periods of timeor until the active agents are encapsulated in the liposome structure ofeach EVs. The excess amounts of active agents are then removed bycentrifugation through a filter that cuts off at a size of about 20 nm,thereby producing the present EV formulation.

Optionally, depending on the nature of the active agent (i.e.,hydrophobicity or hydrophilicity of the active agent), the amount of theactive agent being loaded into the EVs may be improved by the use ofsonication, electroporation, centrifugation, co-solvent, surfactant orlipid.

Alternatively, the present formulation may also be produced by a methodcomprising the steps of,

(a-2) subjecting the mesenchymal stem cell to the treatment of theactive agent or a polynucleotide encoding the active agent; and

(b-2) isolating EVs from the active agent or the polynucleotide encodingthe active agent treated mesenchymal stem cell of step (a-2).

In this embodiment, MSCs are cultured in a medium containing the activeagent (e.g., metformin, lipopolysaccharide, bryostatin or aspirin) orthe polypeptide useful in encoding the active agent for sufficientperiod of time, which allows EVs to be secreted into the supernatant,and subsequently being harvested therefrom. According to the embodimentsof the present disclosure, the harvested EVs comprise the active surfaceproteoglycans, tetraspanins, peptides, growth factors and/or miRNAstherein.

According to certain embodiments, MSCs are cultured underpre-conditional incubation of active agent followed by the treatment ofa hypoxic condition (e.g., 1% 02) for 24-48 hours in a serum-free mediumthat promotes secretion of EVs, so that EVs released therefrom (i.e.,from the cultured MSCs) would have comprised the active agent (e.g.,proteoglycans, proteins, growth factors and/or miRNAs) on and/or insidethe lipid bilayer vesicles.

In the case when the active agent is a peptide (protein) or a RNA (miRNAor siRNA), the peptide/protein aptamers or nucleic acids (e.g. DNAaptamers, RNA aptamers, lipid or glycan affixmers) can be incorporatedinto EVs harvested from MSCs, and alternatively, MSCs may be subject totransfection with a polynucleotide encoding such active agent, so thatthe active agent is expressed in the MSCs. The transfection can be doneany method familiar to the skilled artisan; for example, calciumphosphate co-precipitation, electroporation, nucleofection, cellsqueezing (gently squeezing the cell membrane), sonoporation (inducingpore formation in cell membrane by high-intensity ultrasound), opticaltransfection (generating a tiny hole in cell membrane by highly focusedlaser), impalefection (inserting into a cell DNA bound to the surface ofa nanofiber), gene gun (“shooting” into the cell nucleus DNA coupled toa nanoparticle of an inert solid), magnetofection (using magnetic forceto deliver DNA into target cells), viral transduction (using viruses asa carrier to deliver DNA into target cells), and transfection via adendrimer, a liposome or a cationic polymer. After the transfected MSCswere cultured in a serum-free medium under a hypoxic condition forsufficient periods of time (e.g., 24-48 hours), the EVs secreted intothe supernatant may then be harvested by similar methods describedabove.

Depending on the nature of the active agent to be loaded into the EVliposome, the encapsulation efficacy of the present formulation is about10-90%, preferably about 30-70%, more preferably about 40-50%. Accordingto one specific embodiment, 0.08 to 80 mM of curcumin are respectivelymixed with 0.15 mg/ml EVs isolated from culture supernatants of 1.6×10{circumflex over ( )}7 cells/80 ml, and the encapsulation efficacy isabout 46%-100% (0.01 mM: 46.2%, 0.2 mM: 100%, 1.0 mM: 100%).

Alternatively or optionally, the present formulation may be produced bya method comprising the steps of,

(a-3) irradiating the mesenchymal stem cell with an electronic beam, anX-ray or a gamma ray; and

(b-3) isolating EVs form the electronic beam, X-ray or gamma ray treatedmesenchymal stem cell of step (a-3).

In this embodiment, MSCs are irradiated with a linear accelerator, anX-ray or a gamma ray, which induces MSCs to produce anti-inflammatorymolecules, anti-aging molecules, anti-oxidation molecules and/oranti-radiation molecules. Accordingly, EVs secreted from irradiated MSCswould comprise any of the afore-mentioned molecules within theirliposome structures. According to one specific embodiment, the MSCs areirradiated with X-ray at a dose of 1-10 Gy (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 Gy), followed by the incubation under a hypoxic conditionfor 24-48 hours; the thus-produced EVs are then harvested. Preferably,the MSCs are irradiated with X-ray at a dose of 1-5 Gy. According to oneworking example of the present disclosure, the MSCs are irradiated withX-ray at a dose of 3 Gy.

The formulation produced by any of the methods mentioned above may serveas a biological delivery system, in which the liposome structure of eachEVs acts as a reservoir to carry the active agent (e.g., plantingredient, growth factor, anti-cancer drug, anti-inflammatory molecule,anti-aging molecule, anti-oxidant molecule, anti-irradiation molecule,anti-radiation molecule, vitamin, and/or asta-carotenoid) to a targetcell, and as a shield to protect the active agent encapsulated thereinfrom being rapidly degraded in the bloodstream. Once the presentformulation is in contact with the target cell, the liposome structureof each EVs could then fuse with the cell membrane of the target cell,thereby releasing the encapsulated active agent in the target cell.

According to some embodiments of the present disclosure, the presentformulation is about 20-200 nm in diameter.

According to optional embodiments of the present disclosure, the presentformulation further comprises a helper molecule co-encapsulated orco-embedded in the liposome structure of each isolated EV so as toenhance the therapeutic effect of the active agent. The helper moleculeand the active agent may be co-encapsulated or co-embedded into the EVliposome by use of co-solvent (e.g., PEG 400), surfactant (e.g.,TWEEN-20) or lipid (e.g., LABRAFIL). Preferably, the helper molecule isa cytokine or a defensin.

The cytokine is selected from the group consisting of, granulocytecolony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), interleukin (IL; such as IL-1, IL-2,IL-5, IL-6, IL-8, IL-10, IL-11, IL-12, IL-13, IL-19, IL-20, IL-25, andIL-27), interferon (IFN; such as IFN-α, IFN-β, and IFN-γ), and tumornecrosis factor (TNF; such as TNFα and TNFβ). The defensin isα-defensin, β-defensin or θ-defensin.

According to embodiments of the present disclosure, the helper moleculeand the active agent may be simultaneously or sequentially brought intocontact with MSCs; thus, the EVs subsequently released from the MSCswould comprise both the helper molecule and the active agent in itsstructure. Depending on the nature of the helper molecule, suitablemeans is adopted to bring the helper molecule into contact with MSCs.For example, in the case when the helper molecule is the growth factor,cytokine, defensin or chaperone, it may be introduced into the MSCs viaincubating the MSCs with a culture medium containing the helpermolecule; on the other hand, if the helper molecule is a small molecule(e.g. aspirin or bryostatin), it is brought into contact with MSCs viaits binding receptor or co-receptor and mediate immune regulationfunctions.

Alternatively, the helper molecule and the active agent may besimultaneously or sequentially loaded into an isolated EV via directmixing, sonication, electroporation, centrifugation, gradientcentrifugation, extrusion or the combination thereof, depending on thenature (e.g., hydrophobicity or hydrophilicity) of the helper moleculeand the active agent.

As would be appreciated, the present formulation may be produced bybringing a first molecule into contact with MSCs via an appropriatemethod as described above, followed by mixing the EVs released therefrom(i.e., from the MSCs) with a second molecule via mixing, sonication,electroporation, centrifugation, gradient centrifugation, extrusion orthe combination thereof. According to one embodiment, the first moleculeis the helper molecule and the second molecule is the active agent.According to another embodiment, the first molecule is the active agentand the second molecule is the helper molecule.

(III) Use of the Present Formulation

The third aspect of the present disclosure is directed to the use of thepresent formulation according to any of the above-mentioned embodimentsin treating various diseases, including inflammatory disease, dry eye,degenerative disease, cancer, infectious disease and aging.

According to the embodiments of the present disclosure, the method fortreating a subject in need thereof comprises administering to thesubject an effective amount of the present formulation. The presentformulation may be administered via a route selected from the groupconsisting of, topical, mucosal (e.g. intraconjunctival, intranasal,intratracheal), oral, intraspinal (e.g. intrathecal), intravenous,intraarterial, intramuscular, subcutaneous, intraarticular,intraventrical, intracerebroventricular, intraperitoneal injection andintra-middle ear administration.

For the purpose of treating diseases, about 10⁸ to 10¹⁵ EVs (preferably,10⁹ to 10¹⁴ EVs) of the present formulation, which give rise to about0.1 μg to 1 g of the active agent, are administered to the subject.According to certain embodiments of the present disclosure, the subjectis a mouse, in which about 10¹⁰ to 10¹³ EVs, which give rise to about 10μg to 1 mg of the active agent, are sufficient to produce a therapeuticeffect.

A skilled artisan could calculate the human equivalent dose (HED) of thepresent formulation, based on the doses determined from animal models.As would be appreciated, the actual dosage of the present EV formulationmay be determined by the attending physician based on the physical andphysiological factors of the subject, these factors include, but are notlimited to, age, gender, body weight, body surface, the disease to betreated, severity of the condition, previous history, the presence ofother medications, the route of administration and etc.

According to some embodiments, the present formulation is useful fortreating a subject suffering from an inflammatory disease. The methodcomprises administering to the subject an effective amount of thepresent formulation to regulate the function or expression of immunecells, growth factors, chemokines and/or cytokines. Examples ofinflammatory disease treatable by the present method include, but arenot limited to, psoriasis, colitis, burn injury, acute kidney injury,traumatic brain injury, skin injury, arthritis and autoimmune diseases.

According to certain embodiments, the present formulation is useful fortreating a subject suffering from a degenerative disease. The methodcomprises administering to the subject an effective amount of thepresent formulation to reduce oxidative stress in the subject. In oneembodiment, the oxidative stress is reduced by suppressing therespiratory burst. In another embodiment, the oxidative stress isreduced by increasing the expression of growth factors. In still anotherembodiment, the oxidative stress is reduced by modulating the activationof AMP kinase. Non-limiting examples of degenerative disease treatableby the present method include Parkinson's disease, Alzheimer's disease,dementia, stroke, chronic kidney disease, chronic lung disease orhearing loss.

According to further embodiments, the present formulation is useful fortreating a cancer in a subject. The method comprises administering tothe subject an effective amount of the present formulation so as toinhibit cancer cell growth and/or inhibit cancer cell migration.Examples of the cancer that may be treated by the present methodinclude, but are not limited to, gastric cancer, lung cancer, bladdercancer, breast cancer, pancreatic cancer, renal cancer, colorectalcancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer,hepatocellular carcinoma, melanoma, esophageal carcinoma, multiplemyeloma, and head and neck squamous cell carcinoma.

In certain embodiments of the present disclosure, the presentformulation exhibits an anti-aging effect in the subject. According toone example, the present formulation improves the cognition ability inthe subject.

According to alternative embodiments, the present formulation exhibit atherapeutic effect on spinal cord injury, in which the EV of the presentformulation specifically targets the injured region of spinal cord.

Alternatively, the present formulation is useful in treating aninfectious disease via promoting the direct microbicidal or immunedefense (e.g. cell and humoral immunity) of the subject, in which theinfection is caused by a bacterium, a virus or a fungus.

According to some working examples of the present disclosure, the activeagent additively or synergistically enhances the therapeutic effect ofEV. In certain examples, compared with the EV derived from cancer cells,the EV derived from ucMSCs provides a more efficient means to treatdiseases.

The following Examples are provided to elucidate certain aspects of thepresent invention and to aid those of skilled in the art in practicingthis invention. These Examples are in no way to be considered to limitthe scope of the invention in any manner. Without further elaboration,it is believed that one skilled in the art can, based on the descriptionherein, utilize the present invention to its fullest extent. Allpublications cited herein are hereby incorporated by reference in theirentirety.

Example

Materials and Methods

Cell Culture

The C2C12 myoblasts were purchased from the Global Bioresource Center(ATCC), and cultured in Dulbecco modified eagle medium (DMEM) containing10% FBS. The U937 myeloid cells and DLD-1 cancer cells purchased fromATCC were cultured in RPMI-1640 medium containing 10% FBS. All cellswere incubated at 37° C. in a 5% CO₂ humidity incubator.

Isolation and Characterization of Umbilical Cord Mesenchymal Stem Cell(ucMSC)

A length of 5-cm umbilical cord was taken from an umbilical cord ofnormal spontaneous delivery. The 5-cm cord was put into a normalsaline-containing conical tube and subject to harvest the matrix afterstripping out the umbilical cord vessels. The umbilical cord matrix wascut into pieces about 1 cm in diameter and subject to the culture in adish with low oxygen and low glucose DMEM. The ucMSCs (CD29⁺, CD34⁻,CD44⁺ and CD73⁻′) migrated from the umbilical cord matrix were confirmedby flow cytometry with specified antibodies. The analysis data wasdepicted in FIG. 1.

Isolation and Characterization of Extracellular Vesicles from ucMSC(Hereinafter ucMSC-Naive-EVs)

The ucMSCs were cultured in low glucose DMEM containing 10% FBS. Toisolate the ucMSC-naive-EVs, the culture medium was replaced with theserum-free DMEM; after incubating under a normoxic or hypoxic conditionfor 48 hours, the supernatants were collected, and filtrated by aretention centrifugation (with a 0.02 um filter, the molecular weightcutoff value was about 100 kilodalton) and a 0.20 um filter. TheucMSC-naive-EVs having a diameter ranging between 0.02 to 0.20 um wereisolated and concentrated (200× concentration). The concentrateducMSC-naive-EVs were then analyzed by flow cytometry and westernblotting with anti-CD81 and anti-CD63 antibodies, respectively.

Besides, the growth factors encapsulated in the ucMSC-naive-EV wereanalyzed by multiplex antibody-coated beads array. The data wassummarized in Table 1.

Isolation of EVs Derived from Post-Isolation Incorporation

The hydrophobic curcumin (with cationic character) may be efficientlyencapsulated in the liposome structure of EVs via incubation at roomtemperature and under a condition of pH 6.0-10.0. In the present study,0, 0.02, 0.20 or 1.25 mM curcumin was added to 20 ml ucMSC-naive-EVs(about 1×10¹¹ vesicles/ml) in serum free medium (pH=10.0) followed byincubation at room temperature for 15 minutes. The mixture was thensubject to a 0.02 μm filter so as to obtain the ucMSC-naive-EVs havingcurcumin encapsulated therein (hereinafter EV-curcumin; the finalconcentration was about 1.2 mg/ml). The incorporation efficacy ofcurcumin is 100% to 46.2%. The curcumin concentrations were measured byOD₄₂₀ nm and calculated by a standard curve made with a series ofwell-known concentrations.

Isolation of EVs Derived from Pre-Treated ucMSCs

Thirty culture dishes of ucMSCs, each dish with 4×10⁴ cells/ml in 10 ml,were cultured in low glucose DMEM medium with 10% FBS and pre-treatedwith various small molecules, including aspirin (250 ng/ml) orbryostatin 1 (10 nM) for 1 day. The culture medium was replaced withserum-free low glucose DMEM medium 24 hours later, and the supernatant(300 ml) was collected and filtered by a 0.45 um filter to deplete celldebris, by another 0.2 um filter to cut-off apoptotic bodies, andfinally by a cassette (cartridge) with 0.02 um filter to concentrate theEVs to 1.2 mg/ml so as to respectively obtain the aspirin-EV (7.1×10¹²vesicle/ml) and bryostatin-EV (2.7×10¹² vesicle/ml).

Isolation of EVs from a Linear Accelerator Irradiation Treated ucMSCs(Hereinafter ucMSC-Irradiation-EV)

ucMSCs (4×10⁴ cells/ml in 300 ml) were cultured in low glucose DMEMmedium with 10% FBS for 1 day. Then, the medium were replaced with theserum-free low glucose DMEM medium before the irradiation of 3 Gy(sublethal dose) delivered by a linear accelerator (Clinac 1800, VarianAssociates, Inc., Calif., USA). After the 3 Gy irradiation, the cellswere cultured for another 2 days. The supernatant (300 ml) was collectedand filtered in accordance with the protocols illustrated above.

For the purpose of evaluating the biological function of thethus-obtained ucMSC-irradiation-EVs, ucMSCs (3×10⁴ cells/well) or C2C12myoblasts (5×10⁴ cells/ml) were cultured on a 48-well plate overnight.The serum-free media containing different ucMSC-irradiation-EVs wereadded to the cells followed by irradiating the cells with or without 6Gy delivered by the linear accelerator in a single fraction. One daylater, the reactive oxygen species (ROS) production was assessed by 1 μMdichlorofluorecein hydrochloride (DCFH) followed by the analysis of flowcytometry.

Incubation of EVs and Small Molecules

The polypeptide SIYRRGARRWRKL (also known as ZIP polypeptide, SEQ ID NO:1, m.w. 1982.4, 0.5 uM) was mixed with 0.12 mg/ml ucMSC-naive-EVs (about(2.2×10¹¹ vesicle/ml) dissolved in RPMI medium followed by incubation atroom temperature for 60 minutes. Compared with ucMSC-naive-EVs, theucMSC-naive-EVs comprising the polypeptide (hereinafter EV-ZIP)exhibited higher molecular weight, and accordingly, may be isolated byfiltration (the molecular weight cutoff value was about 100 kilodalton).

For the preparation of EVs having RNA (e.g., miRNA or siRNA)encapsulated therein, 100 ul of ucMSC-naive-EVs (about 10⁹ vesicles, 100ug) were incubated with 2 ug of miRNA-10a (CACAAAUUCGGAUCUACAGGGUA from5′-end to 3′-end; SEQ ID NO: 2) in the transfection solution containing10 ul transfection agent and 40 ul phosphate-buffered saline (PBS)buffer. The mixture was incubated at 37° C. for 10 minutes. The reactionwas stopped by adding 30 ul stop solution (Exoquick) and left on 4° C.ice for 30 minutes. The ucMSC-naive-EVs comprising miRNA10a (hereinafterEV-miRNA10a) was separated with the ucMSC-naive-EVs viaultracentrifugation at 10,000 g for 5 minutes.

Treatment of Muscle Cells

C2C12 myoblasts (1×10³ cells/well) were respectively administered withspecified treatments under serum-free condition for 24 hours. The cellviability and proliferation of the treated C2C12 myoblasts were analyzedby trypan blue exclusion assay and CCK-8 cell proliferation assay.

Treatment of Myeloid Cells

The U937 myeloid cells were seeded in 24-well plate at a density of8×10⁴ cells/well (in 0.4 ml) followed by the stimulation of 100 nM PMA(phorbol myristate acetate) at 37° C., 5% CO2 incubator for 48 hours.The cells were washed with PBS twice before adding medium with andwithout ucMSC-naive-EVs (0.15 mg/ml), curcumin (Cur, 0.8 mM) orEV-curcumin (Cur/EVs, 0.8 mM) for 30 minutes. The reactions wereincubated with DCFH (dichlorofluorescein hydrochloride) at 1 uM for 20minutes before flow cytometric analysis of H₂O₂-mediated fluorescence at480 nm emission.

Analysis of miRNA Profiles in EVs

The EVs (1.0 ml, 2.2×10¹² vesicles/ml) harvested from DLD-1 cells (DEV)or ucMSCs (MEV), and the EVs having curcumin incorporated therein(EV-curcumin) were respectively subject to RNA extraction followed bymiRNA sequencing analysis. In brief, 700 ul of QIAzol Lysis Reagent(Qiagen) was added to EV pellets for homogenenation; the mixture wasincubated at room temperature for 5 minutes, followed by adding 140 ulchloroform and vigorously shaking for 15 seconds. The RNA samples wereharvested by 12,000 g at 4° C. for 15 minutes in accordance with themanufacturer's instruction. Small RNA sequencing (RNA-Seq), includingmicroRNAs (miRNAs) sequencing was done by next generation sequencing(NGS). Small RNA-Seq can query thousands of miRNA sequences withunprecedented sensitivity and dynamic range using TRUSEQ® Small RNA(Illumina) sample preparation protocol. The data were matched to humanshort noncoding RNA sequences (Homo sapiens miRNAs, Hsa miR) andanalyzed with miRBase database.

Analysis of Proteomic Profiles in EVs

The EVs (0.5 ml, 1.1×10¹² vesicles/ml) harvested from ucMSCs (MEV) orDLD-1 cells (DEV), and the EVs having curcumin incorporated therein(EV-curcumin) were respectively subject to a protein lysing buffer. Theprotein concentrations in different EVs were measured by protein assaykit, and equalized proteins from a mixture of 3 batches of samples weresubjected to the ITRAQ™ (isobaric tags for relative and absolutequantification). The ITRAQ™ method is a protein quantitation methodbased on the peptides labelling with a compound that produces isobaricfragments. Employing Applied Biosystems ITRAQ™ Reagents (AppliedBiosystems Inc., USA) provided as a set of four, isobaric (same mass)reagents: ITRAQ™ Reagent 114, ITRAQ™ Reagent 115, ITRAQ™ Reagent 116,and ITRAQ™ Reagent 117 to label the proteins of EVs, we made fourreagents to allow 4-plex samples analysis in a LC/MS-MS experiment. Atotal of 1160 proteins was identified by this ITRAQ™. We define thecomparison ratio over 2 and 4 as a cut-off to show the differentialdisplays.

Effects of EVs on T Cell Differentiation

In response to stimulation, T cells differentiate from resting status(Tho) toward different directions of T cell functions. While the T celldifferentiation into T helper 1 (Th1), T helper 2 (Th2), T regulatory(Treg) or Th17, their cell surface receptors express differentcharacteristics. The CXCR3 expression presents Th1; the CCR4 expressionpresents Th2; CD25 expression presents Treg and CCR6 expression presentsTh17 differentiation. Employing flow cytometric analyses of anti-CXCR3,anti-CCR4, anti-CD25 and anti-CCR6 antibodies (eBiosources Inc. USA),the T cell differentiation was analyzed by modulated with different EVs.In brief, peripheral blood mononuclear cells at 2×105 cells/well in 300ul culture medium were incubated without or without anti-CD3 (10ug/well) and anti-CD28 (2 ug/well) antibodies for 4 days; different EVs(including ucMSC-naive-EV, bryostatin-EV, aspirin-EV or EV-miRNA10a) wasadded into the well containing anti-CD3 and anti-CD28 antibodies on day2 and day 3.

Skin Application of EVs to Treat Psoriasis

In a mouse psoriasis model, a formulation of 62.5 mg imiquimod (5%) ineye cream (187.5 mg) was used to induce mouse (BALB/c mice) psoriasis ofear auricles. The formulation was administered to the ear auricles ofBALB/c mice for three consecutive days. Then, the mice were assignedinto four groups: (A) control group, in which the formulation wasadministered to the ear auricles of mice from day 4 to day 7; (B)EV-treated group, in which the formulation containing 2×10¹¹ vesicles/mgof ucMSC-naive-EVs was administered to the ear auricles of mice from day4 to day 7; (C) EV-curcumin group, in which the formulation containing2×10¹¹ vesicles/mg (about 120 ug protein/mg) EV-curcumin wasadministered to the ear auricles of mice from day 4 to day 7; and (D)curcumin group, in which the formulation containing 1 uM curcumin wasadministered to the ear auricles of mice from day 4 to day 7. Themorphology of ear auricle thickness and vessel inflammation (dilatation)were analyzed on day 14.

Mucosal Application of EVs to Treat Dry Eye

In a mouse model of dry eye, BALB/c mice were maintained at 23° C. underlow humidity of 10% so as to induce the dry eye condition. The mice werealso received escopan 0.15 ml (2.5 mg/ml) subcutaneously twice a day for3 days. Three days after the mice (100%) developed dry eyes, the topicaldrop solution with or without ucMSC-naive-EVs or aspirin-EV at 10 ul(about 12 ug protein of EVs, 2.2×10¹⁰ to 7.1×10¹⁰ vesicles) wasadministered to the eyes of mice for 4 consecutive days. Theconjunctival inflammation was evaluated by administration of 5 ul of 10%fluorescein for 30 seconds and washed with 1 ml PBS under anesthesia.

Parenteral Application of EVs to Treat Hearing Loss

In a mouse model of hearing loss, the inner cell damage was induced byadministering to C57BL/6 mice 4 mg/kg cisplatin daily for 5 consecutivedays, in which the mice were intraperitoneally treated with 0.1 ml PBS(serving as the negative control group), 0.1 ml ucMSC-naive-EVs or 0.1ml EV-curcumin (about 120 ug protein of EVs, 2.2×10¹¹ to 2.8×10¹¹vesicles) from day 1 to day 5. The protecting effect of ucMSC-naive-EVsand EV-curcumin was assessed by analyzing the cell number and functionof the inner ear cells. To assess the protecting effect of the presentEV formulation on hearing loss and inner ear cell death, auditory brainresponse (ABR) threshold at 12 kHz and immunofluorescence images oforgan of Corti explants were measured 10 days post-treatment. To obtainthe ucMSC-EVs preparations with and without curcumin incorporationenough for the animal study requiring a larger amount of EVs for dailyintraperitoneal injection, the ucMSC-EVs in paired formulations with andwithout curcumin incorporation were prepared and stored at −80° C. forat least 3 months. To make sure homogeneous application of ucMSC-EV toeach mouse, different batches of EVs were thawed and mixed for theexperiment right before the animal study. One-way ANOVA with Duncan posthoc test was performed when compared with the control or cisplatingroup.

Parenteral Application of EVs to Treating Aging Mice

In a mouse model of anti-aging, BALB/c mice were subcutaneously treatedwith 100 mg/kg D-galactose every day for 6 consecutive weeks. Then, themice were intraperitoneally administered with 0.1 ml PBS (serving as thenegative control group), 0.1 ml curcumin (1 μM), 0.1 ml ucMSC-naive-EVs,0.1 ml EV-curcumin (about 120 ug protein of EVs, 2.2×10¹¹ vesicles), or5 mg/kg β-lapachone three times a week for 3 weeks after 6 weeks ofaging induction and the pre-test of cognition assessment. The anti-agingeffect of ucMSC-naive-EVs and EV-curcumin was then assessed by thecognition ability in Morris water maze model.

Distribution of EVs in Spinal Cord

For the purpose of investigating the distribution of EVs in spinal cord,1.2 mg/ml (2.2×10¹² vesicles) of ucMSC-naive-EVs were intrathecallyinjected to Sprague-Dawley rats (n=3). In brief, the ratsunder-anesthesia were inserted with a catheter under intrathecal routefor continuous infusion of ucMSC-naive-EVs at a rate of 1 ul per hourfor 72 hours. The ucMSC-naive-EVs (400 ul) were labeled with 20 ulExo-green protein labeling kit (Carboxyfluorescein Succinimidyldiacetate ester-CFSE, EXOG200A-1) at 37° C. for 10 minutes and stop at4° C. for 30 minutes followed by centrifugation at 10,000 g for 5minutes. The labeled ucMSC-naive-EVs were resuspended to 400 ul PBS forstudies.

The rats with unilateral disruption of the dorsal root at L5 level weresacrificed, and the dorsal roots (including the un-injured L4 andinjured L5 dorsal roots) thereof were harvested for tissue section andcounter stained with DAPI (4′,6-diamidino-2-phenylindole). Thedistribution of ucMSC-naive-EVs in spinal cord was detected by afluorescent microscope.

Example 1 Characterization of ucMSC-Naive-EVs

The bio-markers and bio-activity of ucMSC-naive-EVs were examined inthis example, and the results were respectively depicted in Table 1 andFIG. 2.

The flow cytometry and western blotting analysis respectivelydemonstrated that the present ucMSC-naive-EVs exhibited both thetetraspanin CD81 and the EV marker CD63 (FIG. 2, Panel A and B). Thesize (50-150 nm) of the ucMSC-naive-EVs was further confirmed by TEM(FIG. 2, Panel C). The expression level of growth factors inucMSC-naive-EVs was evaluated by multiplex antibody-coated beads array,and the results were summarized in Table 1.

TABLE 1 Growth factors in ucMSC-naive-EVs Growth factors ucMSC-naive-EVDrop through (pg/ml) N Mean SE N Mean SE P values Angiopoietin 6 173.590.5 6 2.2 0.5 0.025 Endoglin 6 87.3 32.0 6 1.9 0.3 0.021 FGF-2 6 57.29.7 6 31.9 0.7 0.021 Follistatin 6 6867.1 2404.7 6 143.8 56.4 0.011 HGF6 11858.0 3141.4 6 48.1 16.7 0.011 IL-8 6 99.2 40.6 6 31.4 17.9 0.033VEGF-C 6 1353.0 185.7 6 77.7 24.4 0.001

The data of Table 1 indicated that various growth factors, includingangiopoietin (a vascular growth factor regulating the angiogenesispathway), endoglin (a subunit of the TGF-β receptor complex thatregulates the angiogenesis and TGF-β signaling pathway), fibroblastgrowth factor-2 (FGF-2, also known as basic fibroblast growth factor; agrowth factor possessing broad mitogenic and cell survival activities),follistatin (also known as activin-binding protein; a growth factorassociated with cellular proliferation, inflammation, folliculogenesis,and development), hepatocyte growth factor (HGF, also known as scatterfactor; a growth factor regulating cell growth, motility, morphogenesis,angiogenesis, tumorigenesis, tissue regeneration, and immuneregulation), interleukin-8 (IL-8; a chemokine inducing chemotaxis ofimmune cells, and regulating immune response), vascular endothelialgrowth factor-C (VEGF-C; a member of the platelet-derived growthfactor/vascular endothelial growth factor (PDGF/VEGF) family that playsa role in lymphangiogenesis, cellular survival, growth, migration,neural development, and blood pressure regulation) were encapsulated inucMSC-naive-EVs.

For the purpose of investigating the bio-activity of EVs, the C2C12myoblasts were treated with different doses of EVs isolated either fromucMSCs or from DLD-1 cancer cells. The data indicated that the treatmentof EVs promoted cell growth in a dose-dependent manner (FIG. 2, PanelD). Further, compared with DLD-1-isolated EVs (designated as DEV), theEVs isolated from ucMSC (designated as MEV) significantly enhanced cellgrowth (P<0.01, n=6; FIG. 2, Panel D).

These results indicated that compared with the control group or EVsisolated from other cells, the present ucMSC-naive-EVs exhibited a moreobvious effect on enhancing cell growth.

Example 2 Characterization of EVs Comprising Different Active AgentsTherein

2.1 Bio-Activity

In addition to ucMSC-naive-EVs, the bio-activity of EVs comprisingcurcumin (i.e., EV-curcumin) or miRNA10a (EV-miRNA10a), or the EVsisolated with pre-conditional treatment of bryostatin (i.e.,bryostatin-EV) or aspirin (i.e., aspirin-EV) was measured in thisexample. The data were depicted in FIGS. 3-4, 7-8 and Table 2.

The expression level of growth factors in ucMSC-naive-EV and EV-curcuminwere analyzed by multiplex antibody-coated beads array, and the resultswere summarized in Table 2.

TABLE 2 Growth factors in ucMSC-naive-EV and EV-curcumin Growth factorsucMSC-naive-EV EV-Curcumin (pg/ml) Means SE Means SE P valuesAngiopoietin 452.1 476.3 402.1 161.2 0.171 Endoglin 169.6 101.2 114.137.9 0.500 FGF-2 25.8 19.8 23.0 4.9 0.150 Follistatin 2993.8 1307.69999.8 2147.9 0.029 HGF 4148.8 2676.0 7224.6 837.1 0.242 IL-8 173.5141.8 2454.3 923.1 0.029 VEGF-C 1425.5 832.7 3230.8 302.2 0.100 Datapresented are calculated from 4 experiments and tested by Mann Whitney Utest.

The data of Table 2 indicated that compared to ucMSC-naive-EVs, theexpression level of follistatin and IL-8 was significantly (p=0.029)higher in EV-curcumin, while the expression level of angiopoietin,endoglin, FGF-2, HGF and VEGF-C in EV-curcumin was similar with that ofucMSC-naive-EVs.

The data of FIG. 3 indicated that different EVs exhibited varied effectson the promotion of C2C12 cell growth, in which aspirin-EVs (Asp-EV)induced a significantly higher growth rate (P<0.05, n=3) thanEV-Curcumin (EV-Cur).

For the purpose of investigating the effect on inflammatory response,U937 myeloid cells were respectively treated with medium only (servingas the negative control group), ucMSC-naive-EV (EVs), curcumin (Cur),and EV-curcumin (Cur/EVs). Compared with the control group, thetreatment of ucMSC-naive-EV, curcumin or EV-curcumin significantlydecreased the production of reactive oxygen species as determined byH₂O₂-mediated fluorescence at 480 nm emission, in which theincorporation of curcumin synergistically enhanced the anti-oxidationeffect of ucMSC-naive-EV (FIG. 4).

In addition, the effects of ucMSC-naive-EV, bryostatin-EV, aspirin-EVand EV-miRNA10a on T cell differentiation were also examined by flowcytometric analysis with antibody specific for CXCR3 (a marker of Th1cells), CCR4 (a marker of Th2 cells), CD25 (a marker of Treg cells), orCCR6 (a marker of Th17 cells). Resting T cells had low level ofdifferentiation marker expressed thereon, and the stimulation withanti-CD3 (10 ug/ml) and anti-CD28 (2 ug/ml) (Ctrl group) for 4 dayssignificantly induced the expression of CXCR3, CCR4 and CD25 (P<0.01,n=6), but not the expression of CCR6 (FIG. 8). Additional administrationof different EVs on day 2 and day 3 showed varied effects on the Th celldifferentiation. The treatment of ucMSC-naive-EVs (EV) significantlyenhanced CD25 and CCR4 expression (*denotes P<0.05, n=6); the dataindicated that ucMSC-naive-EV (EV) treatment induced Treg and Th2differentiation (FIG. 8, Panels B and C). On the other hand, comparedwith bryostatin-EV, the treatment of EV-miRNA10a significantlystimulated CCR4 (# denotes P<0.05, n=6) and CCR6 (*denotes P<0.05, n=6)expression by T (CD4) cells (FIG. 8, Panels B and D). All treatments didnot obviously stimulated CXCR3 expression in comparison with the controlgroup (FIG. 8, Panel A).

These results suggested that the active agent may additively orsynergistically enhance the effect of ucMSC-naive-EVs on different typesof cells and/or diseases.

2.2 Concentration and Size

The sizes and concentrations of different formulations of ucMSC-EVscomprising specified active agent were evaluated by NanoSight NS300nanoparticle tracking analysis. The data of Table 3 and FIG. 7 indicatedthat the vesicles with sizes between 30 and 300 nm ranged between2.2×10¹² and 7.1×10¹² vesicles/ml. The mean of vesicle sizes rangedbetween 151 and 175 nm, and the mode ranged between 107 and 174 nm(Table 3 and FIG. 7).

TABLE 3 Number and size of different specified EVs Number/Size EVEV-curcumin bryostatin EV aspirin-EV Vesicles (10¹²) 2.18 2.79 2.73 7.08Mean (nm) 162.3 151.2 167.2 175.4 Mode (nm) 143.2 106.7 174.1 165.6

Example 3 miRNA and Protein Expression in EVs

3.1 miRNA Expression

Based on over 2000 miRBase database, 36 Hsa miR were matched andidentified, in which 2 miRNA (hsa miR10a-5p and miR182-5p) wereoverexpressed DLD-1-derived EVs (DEV), while the expression levels ofthe other 34 miRNAs were higher in ucMSC-naive-EV (MEV) with and withoutcurcumin incorporation in comparison with the DLD-1-derived EVs (FIG.5). As the data of FIG. 5 depicted, the miRNA profiles ofucMSC-naive-EVs were obviously different from that of DLD-1-derived EVs,in which the incorporation of curcumin increased the expression of 5miRNAs (including 191-5p, 125b-5p, Let-7f-5p, 125a-5p and 127-3p), whiledecreased the expression of 2 miRNAs (including 10a-5p and 21-3p) inEVs.

3.2 Protein Expression

The protein profiles of EVs derived from ucMSCs (i.e., MEV) weredramatically different from that derived from DLD-1 colon cancer cells(i.e., DEV), while the incorporation of curcumin marginally altered theprotein expression (FIG. 6). A total of 1160 proteins was identified byiTRAQ (isobaric tag for relative and absolute quantitation) LC/MS-MSspectrometry, in which only the difference at changes >2 fold or <1/2fold was defined. The protein profiles of specified EVs were summarizedin Table 4.

TABLE 4 Protein profiles of specified EVs EVs DEV/MEVEV-curcumin/MEV >2X Up-expression 135 17 >2X Down-expression 182 34 >4XUp-expression 25 3 >4X Down-expression 29 1

The data of Table 4 indicated that when defining the difference atchanges >2 fold or <1/2 fold, 317 (135+182) proteins were differentbetween ucMSC-naive EVs (MEV) and EVs derived from DLD-1 cancer cells(DEV), and 51 (17+34) proteins are different between MEV and ucMSC-naiveEVs with curcumin incorporation (EV-curcumin). When defining thedifference at changes >4 fold or <1/4 fold, 54 (25+29) proteins weredifferent between ucMSC-naive EVs (MEV) and EVs derived from DLD-1cancer cells (DEV), and only 4 proteins (increases in 3: Ras-relatedprotein Rab-1A, Receptor-type tyrosine-protein phosphatase F, Celladhesion molecule 1; decrease in one: Hepatoma-derived growth factor)were different between MEV and MEV with curcumin incorporation(EV-curcumin) (Table 3). These changes could be implicated in anti-tumoror anti-inflammation treatment.

Example 4 ucMSC-Irradiation-EVs Reduced Reactive Oxygen Species (ROS) byucMSCs

Higher dose of irradiation (6 Gy) significantly increased the level ofROS in ucMSCs (FIG. 9). The data of FIG. 9 further indicated that theucMSC-irradiation-EVs harvested from the precondition of 3 Gyirradiation (Rad-EVs) significantly reduced the ROS production in ucMSCsexposed to a higher dose of irradiation at 6 Gy in four reproducibleexperiments; in contrast, the ucMSC-naive-EVs (EVs) harvested withoutpreconditional irradiation did not exhibit the protecting effect.

Example 5 EV-ZIP Modulated T Cell Differentiation

Resting T (CD4⁺ and CD8⁺ T cells) cells had low CCR4 expressed thereon(Neg group), and the stimulation of anti-CD3 (10 ug/ml) and anti-CD28 (2ug/ml) for 4 days as a positive control (Ctrl group) induced significantincreases in CCR4 expression on CD8 and CD4 cells (P<0.05, n=4) (FIG.10). Additional administration of EVs with or without polypeptideincorporation on day 2 and day 3 showed varied effects on the Th2 celldifferentiation, in which the treatment of ucMSC-EVs (EV) significantlyenhanced CCR4 expression (*denotes P<0.05, n=4), and EV-ZIP (EV-ZIP)significantly suppressed the CCR4 expression on CD8 but not on CD4 cells(# denotes P<0.05, n=4) (FIG. 10).

Example 6 EV-miRNA10a Modulated Cancer Cell Migration

ucMSC-naive-EVs carry its own specific miRNA profiles (FIG. 5), but notcarry all human cell miRNAs. For those under-expression orover-expression of miRNAs in EVs, the antagomir for combating theexpression of over-expressed miRNAs or the agomir for enhancing theexpression of under-expressed miRNAs may be incorporated in EVs so as toimprove the bio-functions of EVs. In this example, the antagomir ofmiRNA10a was incorporated into EVs, and the anti-tumor effect thereofwas evaluated. The data of FIG. 11 demonstrated that the EVs isolatedfrom DLD-1 cancer cells (DEV) enhanced DLD-1 cell migration, and thetreatment of ucMSC-naive-EV (MEV) significantly suppressed DLD-1 cellmigration. Compared with DEV, the incorporation of miRNA10asignificantly suppressed DLD-1 cell migration (FIG. 11).

Example 7 Therapeutic Effect in Animal Study

7.1 Skin Topical Application of EVs for Skin Psoriasis Disease

To evaluate the therapeutic effect of the present EV formulation on skinpsoriasis disease, imiquimod was used to induce psoriasis in a mousemodel followed by the treatment of specified EVs in accordance with theprotocol illustrated in Materials and Methods. The data was depicted inFIG. 12.

As shown in FIG. 12 (Panel A), a set of experiments with representativephoto pictures showed the induction of skin psoriasis-like lesions andeffective treatment responses. The quantified result indicated thatadministrating imiquimod (Vehicle) obviously increased ear thickness ofmice, while the treatment of EV-curcumin significantly reduced thepsoriasis scores (ear auricle thickness) (FIG. 12, Panel B).

7.2 Mucosal Application of EVs for Dry Eye Disease

The effect of the present EV formulation on the treatment of dry eyedisease as investigated in this example. The BALB/c mice were treated inaccordance with the protocol illustrated in Materials and Methods. Theimprovement of dry eyes is assessed by changes of the fluoresceindeposition scores under fluoromicroscopy, and the result was depicted inFIG. 13.

The photographs showed the responses of representative treatments (FIG.13, Panel A). The quantified results depicted that compared with mediumonly (SFM, serving as the negative control), the treatment ofucMSC-naive-EVs (EV) or aspirin-EV (Asp-EV) significantly reduced thedry eye scores (FIG. 13, Panels B and C).

7.3 Parenteral Administration of EVs to Treat Hearing Loss

The therapeutic effect of the present EV formulation on hearing loss wasexamined in this example. The C57BL/6 mice were treated in accordancewith the protocol illustrated in Materials and Methods, and the data wasdepicted in FIG. 14. To obtain the ucMSC-EVs preparations with andwithout curcumin incorporation enough for the animal study requiring alarger amount of EVs for daily intraperitoneal injection, batches ofucMSC-EVs were prepared in paired formulations with and without curcuminincorporation, and stored the paired formulations at −80° C. for atleast 3 months. To make sure homogeneous application of ucMSC-EV to eachmouse, different batches of EVs were thawed and mixed for the experimentright before the animal study. This suggests that the EVs formulationswere stable in frozen storage without anti-freezer.

The administration of cisplatin (Vehicle) induced hearing loss in mice,and the treatment of ucMSC-naive-EVs (EV) and EV-curcumin, but notcurcumin alone, significantly rescued the cisplatin-induced hearing loss(FIG. 14, Panels A and B). The inner ear cells were stained withphalloidin (green), myosin7a (MYO7A, red) and nucleus (DAPI, blue), andthe data demonstrated that compared with control group (Cisplatin), thetreatment of ucMSC-naive-EVs (EV) or EV-curcumin obviously reduced thelevel of cells death caused by cisplatin (FIG. 14, Panel C).

7.4 Intraperitoneal Application of EVs for Anti-Aging Effect

Whether the present EV formulation exhibited an anti-aging effect inanimals was investigated in this example. The mice were treated inaccordance with the protocol illustrated in Materials and Methods, andthe data was depicted in FIG. 15.

The data of Morris water maze demonstrated that the administration ofD-galactose-induced cognition impairment. Specifically, compared withthe naive mice, the mice receiving D-galactose for 6 weeks took asignificantly longer time to reach a safety island in the water pool(FIG. 15, Panel A). The treatment of ucMSC-naive-EVs (DG+EV), Curcumin(DG+Curcumin), EV-Curcumin (DG+EV-curcumin) or β-lapachone(DG+β-lapachone) obviously reduced the time to reach the safety island(FIG. 15, Panel B).

7.5 Distribution of EVs in Spinal Cord

For the purpose of evaluating the use of the present EV formulation inthe treatment of spinal cord injury, the ucMSC-naive-EVs were labeledwith CFSE followed by intrathecally injected into mice as described inMaterials and Methods.

As the data of FIG. 16 depicted, the CFSE-labeled EVs were found in theL5 injured dorsal root ganglion but not in the uninjured L4 dorsal rootganglion. The data shown are one set of the representative experimentwith 3 reproducible experiments. The data suggested that theucMSC-naive-EVs specifically entered the injured (inflammatory) tissuebut not normal tissue.

Taken together, these results demonstrated that the present EVformulation is useful in treating various diseases and/or conditions,including cancers, psoriasis, dry eye, hearing loss, aging, and spinalcord injury.

In conclusion, the present disclosure provides an EV formulation fortreating diseases. The present EV formulation comprises an ucMSC-derivedEV, and one or more active agents (e.g., growth factor, siRNA,polypeptide, small molecule, plant extract, or the combination thereof)encapsulated in the liposome structure of the ucMSC-derived EV viapre-conditional treatment or post-isolation incorporation. According toexamples of the present disclosure, the present EV formulation isprepared between pH=6-10 condition, and could be stably stored under−80° temperature without the addition of anti-freezer. The activeagent(s) may additively or synergistically enhance the therapeuticeffect of the ucMSC-derived EVs, and thus, providing a more efficientmeans to treat diseases.

It will be understood that the above description of embodiments is givenby way of example only and that various modifications may be made bythose with ordinary skill in the art. The above specification, examplesand data provide a complete description of the structure and use ofexemplary embodiments of the invention. Although various embodiments ofthe invention have been described above with a certain degree ofparticularity, or with reference to one or more individual embodiments,those with ordinary skill in the art could make numerous alterations tothe disclosed embodiments without departing from the spirit or scope ofthis invention.

What is claimed is:
 1. A formulation comprising an extracellular vesicle(EV) derived from a mesenchymal stem cell, and an active agentencapsulated in the EV, wherein the mesenchymal stem cell is isolatedfrom Wharton jelly of umbilical cord.
 2. The formulation of claim 1,wherein the active agent is selected from the group consisting of, agrowth factor, an immune-modulating agent, an anti-cancer agent, ananti-inflammatory agent, an anti-infection agent, an anti-aging agent,an anti-oxidant agent, an anti-radiation agent, a nucleic acid and acombination thereof.
 3. The formulation of claim 2, wherein the activeagent is aspirin, bryostatin, metformin, lipopolysaccharide or curcumin.4. The formulation of claim 2, wherein the nucleic acid is a smallinterference ribonucleic acid (siRNA), small hairpin ribonucleic acid(shRNA), or micro-ribonucleic acid (miRNA).
 5. The formulation of claim1, wherein the formulation is prepared by a method comprising, (a)isolating the EV from the mesenchymal stem cell; and (b) mixing theisolated EV of step (a) and the active agent so as to encapsulate theactive agent in the isolated EV.
 6. The formulation of claim 1, whereinthe formulation is prepared by a method comprising, (a) subjecting themesenchymal stem cell to the treatment of the active agent; and (b)isolating the EV from the active agent treated mesenchymal stem cell ofstep (a).
 7. The formulation of claim 1, wherein the formulation isprepared by a method comprising, (a) irradiating the mesenchymal stemcell with a linear accelerator, X-ray or gamma ray; and (b) isolatingthe EV from the irradiated mesenchymal stem cell of step (a).
 8. Theformulation of claim 7, wherein in the step (a), the mesenchymal stemcell is irradiated with the linear accelerator at a dose of 1-10 Gray(Gy).
 9. A method of treating an inflammatory disease, dry eye, cancer,or aging in a subject, comprising administering to the subject aneffective amount of the formulation of claim
 1. 10. The method of claim9, wherein the formulation is prepared by a method comprising, (a)isolating the EV from the mesenchymal stem cell; and (b) mixing theisolated EV of step (a) and the active agent so as to encapsulate theactive agent in the isolated EV.
 11. The method of claim 9, wherein thethe formulation is prepared by a method comprising, (a) subjecting themesenchymal stem cell to the treatment of the active agent; and (b)isolating the EV from the active agent treated mesenchymal stem cell ofstep (a).
 12. The method of claim 9, wherein the formulation isadministered to the subject via a route selected from the groupconsisting of, topical, intraconjunctival, intranasal, intratracheal,oral, intraspinal, intravenous, intraarterial, intramuscular,subcutaneous, intraarticular, intraventrical, intracerebroventricular,intraperitoneal injection and intra-middle ear administration.
 13. Themethod of claim 9, wherein the subject is a human.
 14. The method ofclaim 9, wherein the inflammatory diseases is psoriasis, colitis, burninjury, acute kidney injury, traumatic brain injury, skin injury,arthritis or autoimmune diseases.
 15. The method of claim 9, wherein thecancer is gastric cancer, lung cancer, bladder cancer, breast cancer,pancreatic cancer, renal cancer, colorectal cancer, cervical cancer,ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma,melanoma, esophageal carcinoma, multiple myeloma, or head and necksquamous cell carcinoma.
 16. The method of claim 9, wherein theinfectious disease is caused by a bacterium, virus or fungus.